Accessed from 128.83.63.
20 by nEwp0rt1 on Thu Dec 01 21:53:06 EST 2011
54 〈51〉 Antimicrobial Effectiveness Testing / Microbiological Tests
Table 3. Criteria for Tested Microorganisms
For Category 1 Products
Bacteria: Not less than 1.0 log reduction from
the initial calculated count at 7 days,
not less than 3.0 log reduction from
the initial count at 14 days, and no
increase from the 14 days’ count at
28 days.
Yeast and Molds: No increase from the initial calculated
count at 7, 14, and 28 days.
For Category 2 Products
Bacteria: Not less than 2.0 log reduction from
the initial count at 14 days, and no
increase from the 14 days’ count at
28 days.
Yeast and Molds: No increase from the initial calculated
count at 14 and 28 days.
For Category 3 Products
Bacteria: Not less than 1.0 log reduction from
the initial count at 14 days, and no
increase from the 14 days’ count at
28 days.
Yeast and Molds: No increase from the initial calculated
count at 14 and 28 days.
For Category 4 Products
Bacteria, Yeast, No increase from the initial calculated
and Molds: count at 14 and 28 days.
〈55〉 BIOLOGICAL INDICATORS—
RESISTANCE PERFORMANCE
TESTS
TOTAL VIABLE SPORE COUNT
For paper carrier biological indicators, remove three speci-
mens of the relevant biological indicators from their original
individual containers. Disperse the paper into component fi-
bers by placing the test specimens in a sterile 250-mL cup
of a suitable blender containing 100 mL of chilled, sterilized
Purified Water and blending for a time known to be ade-
quate to achieve a homogeneous suspension. It is not unu-
sual for blending times of 15 minutes or more to be re-
quired for optimal recovery. Transfer a 10-mL aliquot of the
suspension to a sterile, screw-capped 16- × 125-mm tube.
For Biological Indicator for Steam Sterilization, Paper Carrier,
heat the tube containing the suspension in a water bath at
95° to 100° for 15 minutes (heat shock), starting the timing
when the temperature reaches 95°. For Biological Indicator
for Dry-Heat Sterilization, Paper Carrier, and for Biological Indi-
cator for Ethylene Oxide Sterilization, Paper Carrier, heat the
tube containing the suspension in a water bath at 80° to
85° for 10 minutes, starting the timing when the tempera-
ture of the spore suspension reaches 80°. Cool rapidly in an
ice-water bath at 0° to 4°. Transfer two 1-mL aliquots to
suitable tubes, and make appropriate serial dilutions in steril-
ized Purified Water, the dilutions being selected as calculated
to yield preferably 30 to 300 colonies, but not less than 6,
on each of a pair of plates when treated as described below.
Where the biological indicator has a low spore concentra-
tion, it may be necessary to modify the dilution series and
to use more plates at each dilution. Prepare a separate series
Official from May 1, 2012
Copyright (c) 2011 The United States Pharmacopeial Convention. All rights reserved.
USP 35
of plates for each aliquot. Place 1.0 mL of each selected
dilution in each of two 15- × 100-mm Petri dishes. Within
20 minutes, add to each plate 20 mL of Soybean–Casein
Digest Agar Medium that has been melted and cooled to
45° to 50°. Swirl to attain a homogeneous suspension, and
allow it to solidify. Incubate the plates in an inverted posi-
tion at 55° to 60° for Biological Indicator for Steam Steriliza-
tion, Paper Carrier, and at 30° to 35° for Biological Indicator
for Ethylene Oxide Sterilization, Paper Carrier and for Biological
Indicator for Dry-Heat Sterilization, Paper Carrier or at the op-
timal recovery temperature specified by the manufacturer.
Examine the plates after 24 and 48 hours, recording for
each plate the number of colonies; and use the number of
colonies observed after 48 hours to calculate the results.
Calculate the average number of spores per specimen from
the results, using the appropriate dilution factor. The test is
valid if the log number of spores per carrier at 48 hours is
equal to or greater than the log number after 24 hours in
each case. For Biological Indicator for Steam Sterilization, Self-
Contained, aseptically remove the three carriers from the
container, and proceed as directed for Biological Indicator for
Steam Sterilization, Paper Carrier.
For Biological Indicators for Moist Heat, Dry Heat, and Gase-
ous Modes of Sterilization, Nonpaper Carriers, aseptically re-
move the three carriers from their original packaging or
container. Place each carrier in a suitable sterile container
containing 100 mL of chilled Purified Water, and sonicate or
shake on a reciprocal shaker for an appropriate time. Fifteen
minutes or more may be required for optimal recovery. A
previous study should be conducted that ensures that the
recovery method results in at least 50% to 300% recovery
of the labeled spore viable count. Transfer a 10-mL aliquot
of the suspension to a sterile, screw-capped 16- × 125-mm
tube. Heat the tubes containing suspensions of Bacillus
atrophaeus, Bacillus subtilis, and Bacillus coagulans at 80° to
85° for 10 minutes. Heat the tubes containing a suspension
of Geobacillus stearothermophilus at 95° to 100° for 15 min-
utes. Start the timing when the lowest temperature of the
stated temperature ranges is reached. Cool rapidly in an ice-
water bath at 0° to 4°. Transfer two 1-mL aliquots to suita-
ble tubes, and make appropriate serial dilutions in Purified
Water. The selected dilutions should be those that will pref-
erably yield 30 to 300 colonies but not fewer than 6 on
each pair of plates when treated as described below. When
the biological indicator has a low spore concentration, it
may be necessary to modify the dilution series and to use
more plates at each dilution. Prepare a separate series of
plates for each aliquot. Place 1.0 mL of each selected dilu-
tion in each of two 15- × 100-mm Petri dishes. Within 20
minutes add the aliquot to each plate containing 20 mL of
agar that has been melted and cooled to between 45° and
50°. Swirl to attain a homogeneous suspension.
For G. stearothermophilus, B. atrophaeus, B. subtilis, and B.
coagulans, use Soybean–Casein Digest Agar Medium and incu-
bate the plates in an inverted position aerobically at the
following respective temperatures for each microorganism:
55° to 60°, 30° to 35°, and 48° to 52°, or at the optimum
temperature specified by the biological indicator manufac-
turer. Examine the plates after 24 and 48 hours. Record the
number of colonies observed on each plate. Calculate the
average number of spores per carrier from the results, using
the appropriate dilution factor. The test is valid if the log
number of spores per carrier at 48 hours is equal to or
greater than the log number after 24 hours in each case.
For Biological Indicators for Moist Heat, Dry Heat, and Gase-
ous Modes of Sterilization, Liquid Spore Suspensions, using G.
stearothermophilus, B. atrophaeus, B. subtilis, and B. coagulans
as biological indicators, prepare an appropriate serial dilu-
tion of the original spore suspension in chilled Purified Water
contained in a sterile, screw-capped 16- × 125-mm tube,
and proceed with the viable spore count procedures speci-
fied under Biological Indicators for Moist Heat, Dry Heat, and
Gaseous Modes of Sterilization, Nonpaper Carriers.
Accessed from 128.83.63.20 by nEwp0rt1 on Thu Dec 01 21:53:06 EST 2011
USP 35 Microbiological Tests / 〈55〉 Biological Indicators—Resistance Performance Tests 55
D-VALUE DETERMINATION
Conduct all the tests described in this section under asep-
tic conditions, using sterilized equipment for nonthermo-
philic microorganisms. D-value determination for G.
stearothermophilus and B. coagulans can be performed in a
controlled but unclassified environment.
Apparatus
The test equipment for the determination of microbial re-
sistance is described in substantial detail in ISO 18472, Ster-
ilization of Health Care Products—Biological and Chemical In-
dicators—Test Equipment.1 The details of individual Biological
Indicator Evaluation Resistometers (BIERs) vary with the spe-
cifics of their design and the particular sterilization process
in conjunction with which they are used. Provided that the
performance of the BIER vessel meets the requirements of
the ISO standard for exposure of the biological indicator,
design differences are acceptable.
Procedure
Carry out the tests for D value at each of the applicable
sets of sterilization conditions for which the packaged bio-
logical indicator under test is labeled for use. Take a suffi-
cient number of groups of specimens of biological indicators
in their original individual containers, each group consisting
of not less than 5 specimens. The number of groups pro-
vides a range of observations from not less than one labeled
D value below the labeled survival time through not less
than one labeled D value above the labeled kill time. Place
each group on a separate suitable specimen holder that per-
mits each specimen to be exposed to the prescribed steril-
izing condition at a specific location in the sterilizing cham-
ber of the BIER. Check the BIER apparatus for operating
parameters using specimen holders without specimens. Se-
lect a series of sterilizing times in increments from the short-
est time for the specimens to be tested. The differences in
sterilizing times over the series are as constant as feasible,
and the difference between adjacent times is no greater
than 75% of the labeled D value.
Test procedures for the use of BIER vessels for the evalua-
tion of microbial resistance are defined in a series of ISO
standards under the 11138 series.2, 3, 4, 5 The appropriate
standard should be followed for the biological indicator. The
test methods and carriers used with the BIER may be
adapted to the specifics of the biological indicator. The
method and apparatus used for paper carriers may differ
from those for other carriers and will be substantially differ-
ent from those used for suspensions of biological indicators.
The D-value exposure conditions for alternative material
carriers are the same as the conditions used to determine
the D value for paper carriers. If the manufacturer’s label
permits usage of the biological indicator carrier with multi-
ple sterilization methods, then data on D value, survival
time, and kill time will need to be provided by the manufac-
1ANSI/ AAMI/ ISO 18472:2006, Sterilization of Health Care Products—Biologi-
cal and Chemical Indicators—Test Equipment. Association for the Advance-
ment of Medical Instrumentation (AAMI), 1110 N. Glebe Road, Suite 220,
Arlington, VA 22201-4795
2ANSI/AAMI/ISO 11138-1:2006, Sterilization of health care products—Biologi-
cal indicators—Part 1: General requirements, 2nd ed. Association for the Ad-
vancement of Medical Instrumentation (AAMI), 1110 N. Glebe Road, Suite
220, Arlington, VA.
3ANSI/AAMI/ISO 11138-2:2006, Sterilization of health care products—Biologi-
cal indicators—Part 2: Biological indicators for ethylene oxide sterilization
processes, 3rd ed. Association for the Advancement of Medical Instrumenta-
tion (AAMI), 1110 N. Glebe Road, Suite 220, Arlington, VA.
4ANSI/AAMI/ISO 11138-3:2006, Sterilization of health care products—Biologi-
cal indicators—Part 3: Biological indicators for moist heat sterilization
processes. Association for the Advancement of Medical Instrumentation
(AAMI), 1110 N. Glebe Road, Suite 220, Arlington, VA.
5ANSI/AAMI/ISO 11138-4:2006, Sterilization of health care products—Biologi-
cal indicators—Part 4: Biological indicators for dry heat sterilization processes.
Association for the Advancement of Medical Instrumentation (AAMI), 1110 N.
Glebe Road, Suite 220, Arlington, VA.
Official from May 1, 2012
Copyright (c) 2011 The United States Pharmacopeial Convention. All rights reserved.
turer for each sterilization method. It is possible that biologi-
cal indicators inoculated onto carriers other than paper will
be used for gaseous or vapor sterilization/decontamination
methods such as vapor phase hydrogen peroxide and chlo-
rine dioxide.
Standard physical conditions for the evaluation of biologi-
cal indicators for use with vapor phase hydrogen peroxide
or chlorine dioxide have not been defined. In the case of
chlorine dioxide, concentration of the gas, relative humidity,
and temperature are critical process control conditions that
can be accurately measured. The manufacturer of biological
indicators marketed for use with chlorine dioxide should
state the conditions under which the D-value determination
was conducted so that the user can at least discern the
resistance of a lot of biological indicators as compared to
their own anticipated use conditions. The situation with va-
por phase hydrogen peroxide is a more complex one. Vari-
ous equipment manufacturers have proposed different de-
contamination or sterilization conditions. Thus, there is no
standard process for the conduct of vapor phase hydrogen
decontamination or surface sterilization. It follows, then,
that there are no industry standard biological indicator eval-
uation methods for vapor hydrogen peroxide, and it has
been reported that there may not be a direct correlation
between vapor concentration and rate or even effectiveness
of biological indicator inactivation. Additionally, it is difficult
to accurately assess relative humidity, which is often defined
as a critical process parameter, in the presence of vapor hy-
drogen peroxide. For these reasons it is more reasonable to
consider resistance of biological indicators to be a relative or
comparative measure from the manufacturer rather than a
true D value. It follows that, depending upon equipment
and processes employed, it may be impossible for an end
user to duplicate the biological indicator resistance tests per-
formed by the manufacturer.
For Biological Indicators for Moist Heat, Dry Heat, and Gase-
ous Modes of Sterilization, Liquid Spore Suspensions, conduct
D-value determinations for each of the microorganisms that
are provided as a liquid spore crop suspension. The test is
conducted using appropriate serial dilutions predicated
upon the stated spore titer of the suspension in Purified
Water in a sterile tube.
Where the suspension is placed on or in a substrate such
as an elastomeric closure or formulated product, its resis-
tance may differ from that determined in Purified Water.
That difference may be significant to the usage of the bio-
logical indicators and appropriate measurements made prior
to use in sterilization validation activities.
Recovery
After completion of the sterilizing procedure for Biological
Indicator for Dry-Heat Sterilization, Paper Carrier; Biological In-
dicator for Ethylene Oxide Sterilization, Paper Carrier; or Bio-
logical Indicator for Steam Sterilization, Paper Carrier, which-
ever is applicable, and within a noted time not more than 4
hours, aseptically remove and add each strip to a suitable
medium (see Media under Sterility Tests 〈71〉) to submerge
the biological indicator completely in a suitable tube. For
each Biological Indicator for Steam Sterilization, Self-Contained
specimen, the paper strip is immersed in the self-contained
medium according to manufacturers’ instructions, within a
noted time not more than 4 hours. Incubate each tube at
the optimal recovery temperature specified by the manufac-
turer. Observe each inoculated medium-containing tube at
appropriate intervals for a total of 7 days after inoculation.
(Where growth is observed at any particular observation
time, further incubation of the specimen(s) concerned may
be omitted.) Note the number of specimens showing no
evidence of growth at any time.
For Biological Indicators for Moist Heat, Dry Heat, and Gase-
ous Modes of Sterilization, Nonpaper Carriers, recovery of
spores from the biological indicator carriers will follow re-
covery procedures described in the procedures under Total
 Accessed from 128.83.63.20 by nEwp0rt1 on Thu Dec 01 21:53:06 EST 2011
56 〈55〉 Biological Indicators—Resistance Performance Tests / Microbiological Tests
Viable Spore Count. D-value determination methods for pa-
per carrier biological indicators may be used to calculate the
D value for nonpaper carriers. Incubation conditions for the
microorganisms that may be used for nonpaper biological
indicators are described in the Total Viable Spore Count
section.
For Biological Indicators for Moist Heat, Dry Heat, and Gase-
ous Modes of Sterilization, Liquid Spore Suspensions, the
method of recovery following sterilization exposure condi-
tions are those methods described in the Total Viable Spore
Count section for liquid suspensions, and when a dry heat
D-value determination is made from B. atrophaeus suspen-
sions, the same recovery procedures as described under Bio-
logical Indicator for Steam Sterilization, Paper Carrier are
followed.
Where C. sporogenes is used as a biological indicator,
methods for preparation, inoculation, and recovery methods
and media must be adapted to accommodate the use of
this anaerobic sporeformer.
Calculation
The determination of D values of biological indicators can
be performed using the Limited Spearman-Karber, Survival
Curve Method or Stumbo-Murphy-Cochran procedures.6, 7, 8
It is preferable to use the same method as that defined by
the biological indicator manufacturer to determine D values.
The use of a different method can result in differences that
are more an artifact of the method than a variation in the
performance of the biological indicator.
Survival Time and Kill Time
Take two groups, each consisting of 10 specimens of the
relevant biological indicator, in their original, individual con-
tainers. Place the specimens of a group in suitable specimen
holders that permit each specimen to be exposed to the
sterilizing conditions at a specific location in the BIER
chamber.
Expose the specimens for the required survival time, enter
the chamber, and remove the holder(s) containing the 10
specimens. Repeat the above procedure immediately, or
preheat if a substantial interval has elapsed, so as to subject
the second holder(s) containing 10 specimens similarly to
the first conditions, but for the required kill time.
The Survival time and kill time for all monographed biolog-
ical indicators is described in the official monograph under
the heading for each.
6 Pflug, I.J. Syllabus for an Introductory Course in the Microbiology and Engineer-
ing of Sterilization Processes, 4th ed. St. Paul, MN: Environmental Sterilization
Services, 1980.
7 Pflug, I.J., and G.M. Smith. The Use of Biological Indicators for Monitoring
Wet-Heat Sterilization Processes, in Sterilization of Medical Products, ed. E.R.L.
Gaughran and K. Kereluk. New Brunswick, NJ: Johnson and Johnson, 1977,
193–230.
8 Holcomb, R.G., and I.J. Pflug. The Spearman-Karber Method of Analyzing
Quantal Assay Microbial Destruction Data, in Microbiology and Engineering
Sterilization Processes, ed. I.J. Pflug. St. Paul, MN: Environmental Sterilization
Services, 1979.
Official from May 1, 2012
Copyright (c) 2011 The United States Pharmacopeial Convention. All rights reserved.
USP 35
〈61〉 MICROBIOLOGICAL
EXAMINATION OF NONSTERILE
PRODUCTS: MICROBIAL
ENUMERATION TESTS
INTRODUCTION
The tests described hereafter will allow quantitative enu-
meration of mesophilic bacteria and fungi that may grow
under aerobic conditions.
The tests are designed primarily to determine whether a
substance or preparation complies with an established speci-
fication for microbiological quality. When used for such pur-
poses, follow the instructions given below, including the
number of samples to be taken, and interpret the results as
stated below.
The methods are not applicable to products containing
viable microorganisms as active ingredients.
Alternative microbiological procedures, including auto-
mated methods, may be used, provided that their equiva-
lence to the Pharmacopeial method has been demonstrated.
GENERAL PROCEDURES
Carry out the determination under conditions designed to
avoid extrinsic microbial contamination of the product to be
examined. The precautions taken to avoid contamination
must be such that they do not affect any microorganisms
that are to be revealed in the test.
If the product to be examined has antimicrobial activity,
this is, insofar as possible, removed or neutralized. If inac-
tivators are used for this purpose, their efficacy and their
absence of toxicity for microorganisms must be
demonstrated.
If surface-active substances are used for sample prepara-
tion, their absence of toxicity for microorganisms and their
compatibility with any inactivators used must be
demonstrated.
ENUMERATION METHODS
Use the Membrane Filtration method or one of the Plate-
Count Methods, as directed. The Most-Probable-Number
(MPN) Method is generally the least accurate method for
microbial counts; however, for certain product groups with
very low bioburden, it may be the most appropriate
method.
The choice of a method is based on factors such as the
nature of the product and the required limit of microorgan-
isms. The method chosen must allow testing of a sufficient
sample size to judge compliance with the specification. The
suitability of the chosen method must be established.
GROWTH PROMOTION TEST, SUITABILITY
OF THE COUNTING METHOD AND NEGATIVE
CONTROLS
General Considerations
The ability of the test to detect microorganisms in the
presence of product to be tested must be established.